Abstract: Bovine mastitis is a frequent cause of economic loss in dairy herds. The Staphylococci are the predominant pathogen in subclinical and chronic bovine mastitis. Coagulase-Negative Staphylococci (CNS) have been isolated from milk samples collected from cows with clinical and subclinical mastitis in several countries, cause tissue damage and decreases in milk production. The purpose of this study was to determine the prevalence and in vitro antibiotic susceptibility of CNS isolated from bovine mastitis in Iran to several antimicrobial agents used in the control of this disease. In this study, 300 bovine milk samples with Intramammary Infections (IMIs) were collected from dairy herds in Mashhad, Iran over one year study period. From a total of 300 milk samples collected, 67 (22.33%) were positive for coagulase-negative staphylococci. CNS strains were identified as 18 (26.86%) strains of S. hyicus, 14 (20.89%) strains of S. chromogenes, 15 (22.39%) strains of S. epidermidis, 10 (14.92%) strains of S. haemolyticus, 3 (4.48%) strains of S. sciuri, 4 (5.97%) strains of S. simulans and 3 (4.48%) strains of S. xylosus. The antibiotic susceptibility test results showed that all CNS strains were susceptible to gentamycin (100%), while they were resistant to penicillin (95.52%), oxacillin (73.14%), sulfamethoxazole/trimethoprim (28.36%), erythromycin (22.39%), tetracycline (23.88%), kanamycin (14.92%), amoxycillin+clavulanic acid (14.92%), oxytetracycline (16.42%) and enrofloxacin (7.46%). Results of this study showed that coagulase-negative staphylococcus is emerging as important minor pathogens and can be the cause of substantial economic losses. The high resistance to penicillin found in this study emphasizes the importance of the identification of coagulase-negative staphylococcus when mastitis is present.